Application of populus tomentosa IPT5b gene in creation of high-yield shade-tolerant populus germplasm

Abstract

The invention discloses an application of a populus tomentosa IPT5b gene in creating a high-yield shade-tolerant populus germplasm, belongs to forest breeding, and is characterized in that a phloem specific promoter CLE41b is utilized to express a cytokinin synthetic gene IPT5b in populus tomentosa, and an overexpression material of which cambium activity and xylem development are remarkably promoted is created. After shading treatment, the number of cambium cell layers and the number of xylem cell layers of CLE41bpro: IPT5b are remarkably higher than those of wild type CLE41bpro: IPT5b, and the phenotype increases the content of cytokinin (CK) in stems, so that the wood yield of trees under the shading condition can be increased. According to the method, a high-yield and shade-tolerant new poplar variety is created in a manner of finely regulating and controlling the content of CK in the phloem of the poplar. The gene source and scientific reference are provided for improving and improving the yield and shade tolerance of other trees, and the gene has important application value in the field of forest tree gene engineering.

Description

Technical Field

[0001] The invention belongs to the field of forest tree breeding, and specifically relates to the application of Populus tomentosa IPT5b gene in creating high-yield shade-tolerant poplar germplasm. Background Art

[0002] Poplar is one of the fastest-growing timber species with the largest cultivated area in the mid-latitude plains in the world. It has the characteristics of fast growth, high yield and easy renewal, and has important economic value and ecological benefits. In natural forests and artificial forests, high stand density will induce trees to compete for light sources, nutrients and water, resulting in plant growth stagnation. Light not only provides energy for plant photosynthesis, but also acts as an important signal factor to regulate plant growth and development. Plants usually perceive shade signals, that is, changes in light quality in the environment (low red light / far-red light ratio) and produce a series of shade avoidance reactions, such as increased plant height, reduced branches, upward leaves, and reduced leaf area. These reactions are collectively referred to as shade avoidance reactions. Shading can also inhibit the activity of the vascular cambium and the development of secondary xylem in the stems of trees, resulting in reduced wood yield. Therefore, creating high-yield, shade-tolerant and densely planted fast-growing forest germplasm is an important way to solve my country's wood supply and demand problems.

[0003] The formation of wood is essentially the development process of secondary xylem. This process specifically involves four steps: the division and differentiation of vascular cambium cells, the expansion of xylem cells, the deposition of secondary wall (SCW), and programmed cell death (PCD). So far, predecessors have made significant progress in understanding the key regulatory factors of these developmental stages of poplar. A variety of plant hormones, including auxin, cytokinin (CK) and gibberellin (GA), synergistically regulate the activity of vascular cambium. Among them, CK is mainly accumulated in the secondary phloem, acting as a local and long-distance signal to regulate the activity of vascular cambium. Studies have shown that overexpression of Arabidopsis thaliana cytokinin oxidase 2 (AtCKX2), a cytokinin decomposition gene, specifically in the cambium or phloem of poplar will lead to reduced CK levels and a reduction in the number of cambium cells in the plant. In contrast, overexpression of Arabidopsis thaliana ISOPENTYLTRANSFERASE 7 (AtIPT7) or LONELY GUY 1 (LOG1), two genes encoding cytokinin biosynthetic enzymes, in poplar increased endogenous CK content and promoted poplar cambium activity and wood formation. However, whether it is possible to create shade-tolerant (low red / far-red ratio) forest germplasm by expressing cytokinin biosynthesis genes remains unclear. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for creating high-yield shade-tolerant poplar germplasm.

[0005] The technical solution of the present invention is: application of the Populus tomentosa IPT5b gene in regulating poplar biomass, cambium activity and / or xylem development, and the nucleotide sequence of the Populus tomentosa IPT5b gene is shown in SEQ ID NO.1.

[0006] Furthermore, the regulation method is: overexpressing the Populus tomentosa IPT5b gene in poplars, thereby increasing the biomass, cambium activity and / or xylem development of the poplars.

[0007] Furthermore, the method for overexpressing the Populus tomentosa IPT5b gene is: using the Populus tomentosa CLE41b gene promoter to drive the overexpression of the Populus tomentosa IPT5b gene, and the nucleotide sequence of the Populus tomentosa CLE41b gene promoter is shown in SEQ ID NO.2.

[0008] A method for creating high-yield, shade-tolerant poplar germplasm, comprising the following steps:

[0009] (1) Construct an overexpression vector of the IPT5b gene of Populus tomentosa driven by the CLE41b gene promoter of Populus tomentosa;

[0010] (2) transforming poplar with the overexpression vector constructed in step (1);

[0011] (3) Tissue culture and screening of positive transgenic plants;

[0012] (4) Planting under natural light and shade conditions, selecting strains with significantly improved biomass, cambium activity and / or xylem development;

[0013] The nucleotide sequence of the promoter of the CLE41b gene of Populus tomentosa is shown in SEQ ID NO.2, and the nucleotide sequence of the IPT5b gene of Populus tomentosa is shown in SEQ ID NO.1.

[0014] Furthermore, the specific method of step (2) is: using the Agrobacterium-mediated leaf disc method to transform the overexpression vector constructed in step (1) into wild-type leaves of poplar.

[0015] An overexpression vector contains a Populus trichocarpa CLE41b gene promoter and a Populus tomentosa IPT5b gene. The nucleotide sequence of the Populus trichocarpa CLE41b gene promoter is shown as SEQ ID NO.2, and the nucleotide sequence of the Populus tomentosa IPT5b gene is shown as SEQ ID NO.1.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention uses the phloem-specific promoter CLE41b to express the cytokinin synthesis gene IPT5b in Populus tomentosa, creating an overexpression material that significantly promotes the cambium activity and xylem development. pro :IPT5b has significantly higher cambium cell layers and xylem cell layers than the wild type, and the phenotype increases the cytokinin (CK) content in the stem, which can increase the wood yield of trees under shade conditions. The present invention creates a new high-yield, shade-tolerant poplar variety by finely regulating the CK content in the poplar phloem. The present invention provides a gene source and scientific reference for improving and increasing the yield and shade tolerance of other trees, and has important application value in the field of forest genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 .Populus tomentosaCLE41b pro : Creation of IPT5b overexpression materials and phenotypic analysis. (A) qPCR detection of wild type (WT) and CLE41b pro : Analysis of the expression level of IPT5b gene in stems of IPT5b overexpressing strains. Different letters represent significant differences (P<0.05, ANOVA analysis). (B) WT and CLE41b pro :Comparison of stem diameter at internode 8 in IPT5b overexpressing plants (L1, L2). (C) WT and CLE41b pro :Comparison of fresh weight of aboveground tissues in IPT5b overexpressing plants (L1, L2). *(P<0.05) indicates Student's t-test analysis.

[0019] Figure 2 .Under white light (WL) and shade treatment (WL+FR), wild type (WT) and CLE41b pro : Phenotypic observation of IPT5b overexpressing plants. (A) Phenotypic diagram of the whole plant, Bar=22 cm; (B) Statistical analysis of plant height, different letters represent significant differences (P<0.05, ANOVA analysis).

[0020] Figure 3 .Under white light (WL) and shade treatment (WL+FR), wild type (WT) and CLE41b pro: Sections and phenotypic analysis of the 7th internode of IPT5b overexpressing plants (L1 and L2). (A) The upper figure shows the phenotype of the number of cambium cell layers in the stem (red dots represent cambium cells), Bar = 50 μm; the lower figure shows the distribution ratio of the number of cambium cell layers in each row (B) Statistical analysis of the number of cambium cell layers, different numbers of characters represent significant differences (p < 0.05, ANOVA analysis); (C) Statistical analysis of the number of xylem cell layers, different numbers of characters represent significant differences (p < 0.05, ANOVA analysis). (D) Comparison of xylem phenotypes in the 7th internode, Xy: xylem. Bar = 100 μm. DETAILED DESCRIPTION

[0021] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0022] (1)CLE41b pro Construction of IPT5b overexpressing plants

[0023] First, the promoter sequence (SEQ ID NO.2) of the CLE41b gene (Potri.002G241300) of Populus trichocarpa was downloaded from the Phytozome database, and specific primers were designed based on the Populus trichocarpa genome information. The Populus trichocarpa genomic DNA was used as a template and the promoter of the CLE41b gene of Populus trichocarpa was amplified using a high-fidelity enzyme. At the same time, the pCXGUS-P plasmid was cut with BamHI and SacI to remove the GUS reporter gene. The promoter of the CLE41b gene was then connected to the pCXGUS-P plasmid after restriction digestion by homologous recombination to construct pCX-CLE41b. pro carrier.

[0024] Subsequently, the coding sequence of the IPT5b gene of Populus tomentosa was downloaded from the Phytozome database, and gene-specific primers were designed based on it. The coding sequence of the IPT5b gene (SEQ ID NO.1) was amplified using the cDNA of Populus tomentosa as a template using a high-fidelity enzyme and inserted into pCX-CLE41b pro Vector (CLE41b pro promoter sequence), and obtained pCLE41b pro :IPT5b overexpression vector; after the vector was verified by sequencing, it was transformed into Agrobacterium.

[0025] (2) Genetic transformation and genotype identification of poplar

[0026] Populus tomentosa Carrière was used as the recipient material and the successfully constructed CLE41b was transferred to the leaf disc method mediated by Agrobacterium. pro:IPT5b overexpression vector was introduced into poplar leaves, and positive transgenic plants with corresponding resistance were screened through poplar tissue culture.

[0027] First, build the successfully CLE41b pro :IPT5b overexpression vector was transferred into Agrobacterium, cultured in a 28℃ incubator for 2 days, and then single clones were selected and cultured in kanamycin-resistant YEP medium at 28℃ shaking overnight. Agrobacterium cultured overnight was added to kanamycin-resistant YEP medium at a ratio of 1:100, and cultured in a 28℃ shaking incubator until OD 600 nm To 0.4-0.6. Then centrifuge in a 4℃ low-temperature centrifuge, 4000rpm, 10min, collect the bacteria, resuspend the bacteria with resuspension solution, add resuspension solution to 30mL, and shake in a 28℃ shaker for 45min. Then cut the poplar leaves into 4-6mm leaf discs, infect them in Agrobacterium solution for 10min, and shake every 3-5 minutes. After 10min, use sterile paper to absorb the bacterial solution on the surface of the leaf disc, then place it on the co-cultivation medium, culture it in the dark for 2 days, and then transfer it to a medium with hygromycin resistance for culture.

[0028] After obtaining positive plants with resistance, we extracted total RNA from plant leaves and obtained cDNA through reverse transcription. We used IPT5b-specific quantitative primers to perform qPCR expression analysis to identify transgenic lines. The specific results are: CLE41b pro :IPT5b L1 expression was upregulated 45-fold, CLE41b pro :IPT5b L2 expression was upregulated 33.6-fold, CLE41b pro :IPT5bL3 expression was upregulated 41.6-fold, CLE41b pro :IPT5b L4 expression was upregulated by 9.2-fold ( Figure 1 (A).

[0029] (3) Phenotypic observation of transgenic plants

[0030] After 10 weeks of growth under normal white light conditions, CLE41b pro :IPT5b overexpressing lines (L1, L2) had larger leaves, thicker stems, and higher aboveground biomass ( Figure 1 In B, C). To compare WT and CLE41b pro :IPT5b strains respond to shade treatment. We compared poplar WT and CLE41b pro IPT5b overexpressing plants (L1 and L2) were cultured under normal white light conditions (R / FR = 10.69) for 10 weeks and then transferred to simulated shade conditions (R / FR = 0.46) for 2 weeks. The control group continued to be cultured under white light. Figure 2 Middle A and Figure 2 As shown in B, CLE41b under white light conditions or shade conditions pro There was no significant difference in plant height between IPT5b overexpressing plants and WT plants.

[0031] Subsequently, we compared WT and CLE41b pro : The 7th internode (counted from the apical bud) of the stem of IPT5b overexpressing plants was sectioned and stained with toluidine blue. Under white light (WL) conditions, CLE41b pro :IPT5b overexpressing plants had an increased number of cambium cell layers and maintained a higher number of cambium layers under shade (WL+FR) conditions ( Figure 3 In addition, CLE41b pro : The xylem development of IPT5b overexpressing plants was also significantly enhanced, and they also had more wood cell layers under shade conditions ( Figure 3 These results indicate that phloem-specifically expressed IPT5b can effectively alleviate the inhibitory effect of shading on plant secondary growth and promote the activity of the plant's cambium and xylem development. This provides a new gene source for regulating tree shade tolerance, cambium activity, and xylem development in the field of forest genetic engineering, and has great application prospects.

Claims

1. Application of the IPT5b gene of Populus tomentosa in regulating poplar biomass, cambium activity and / or xylem development, wherein the nucleotide sequence of the IPT5b gene of Populus tomentosa is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The regulation method is: over-expressing the Populus tomentosa IPT5b gene in poplars, thereby increasing the biomass, cambium activity and / or xylem development of the poplars.

3. The use according to claim 2, characterized in that: The method for overexpressing the Populus tomentosa IPT5b gene is: using the Populus tomentosa CLE41b gene promoter to drive the overexpression of the Populus tomentosa IPT5b gene, and the nucleotide sequence of the Populus tomentosa CLE41b gene promoter is shown in SEQ ID NO.

2.

4. A method for creating high-yield, shade-tolerant poplar germplasm, characterized in that: The steps include: (1) Construct an overexpression vector of the IPT5b gene of Populus tomentosa driven by the CLE41b gene promoter of Populus tomentosa; (2) transforming poplar with the overexpression vector constructed in step (1); (3) Tissue culture and screening of positive transgenic plants; (4) Planting under natural light and shade conditions, selecting strains with significantly improved biomass, cambium activity and / or xylem development; The nucleotide sequence of the promoter of the CLE41b gene of Populus tomentosa is shown in SEQ ID NO.2, and the nucleotide sequence of the IPT5b gene of Populus tomentosa is shown in SEQ ID NO.

1.

5. The method according to claim 4, characterized in that The specific method of step (2) is: using the Agrobacterium-mediated leaf disc method to transform the overexpression vector constructed in step (1) into wild-type leaves of poplar.

6. An overexpression vector, characterized in that: The invention contains a Populus trichocarpa CLE41b gene promoter and a Populus tomentosa IPT5b gene. The nucleotide sequence of the Populus trichocarpa CLE41b gene promoter is shown as SEQ ID NO.2, and the nucleotide sequence of the Populus tomentosa IPT5b gene is shown as SEQ ID NO.1.

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